TGFβ Antibody Partially Reduced the Amount of Scarring
Evidence demonstrates that wound healing is regulated by a group of cytokines, growth factors and their receptors (5-7). They influence cell migration, growth and proliferation in a complex, orchestrated manner and are involved in neutrophil and macrophage infiltration, angiogenesis, fibroplasia, matrix deposition, scarring and reepithelialization. Besides platelets and macrophages, fibroblasts are the major cellular source of cytokines or growth factors during wound healing. The scarless wound healing in fetal skin at early gestation is a result of the unique cytokine or growth factor profile.
Of these, transforming growth factor-beta (TGFβ3) has been most widely studied as it is implicated in the transition between scarless healing and repair with scar formation. Called growth factors for historical reasons, their main function is to control cell proliferation and differentiation and to stimulate the synthesis of extracellular matrix such as collagen. TGFβ has been found by immunohistochemistry in unwounded fetal skin, and high levels of TGFβ are expressed at gestational ages associated with scarless repair. Exogenous application of TGFβ to normally scarless fetal wounds resulted in scar formation and an adult-like inflammatory response was observed. The profibrotic nature of TGFβ was confirmed in wounds of adult rats as a neutralizing TGFβ antibody partially reduced the amount of scarring. TGFβ stimulates collagen I production, which is the predominant collagen type in adult skin. On the other hand, TGFβ neutralizing antibodies do not entirely prevent scarring in the adult skin, and recent studies question the efficacy of TGFβ as a dominant scar-forming factor (8-15).
Studies have also found that decreased and rapidly cleared TGFβ1 and TGFβ2 expression accompanied by increased and prolonged TGFβ3 levels in wounded E16 animals correlated with organized collagen deposition. In contrast, increased and prolonged TGFβ1 and TGFβ2 expression accompanied by decreased and delayed TGFβ3 expression in wounded E19 animals correlated with disorganized collagen architecture. This means that increased TGFβ1, TGFβ2, and decreased TGFβ3 expression is responsible for the late gestation fetal scar formation.
COX-2 Inhibitor Reduces Scar Tissue Formation and Enhances Tensile Strength
While the interleukins IL-6, IL-8, and IL-10 have been studied in fetal wound repair, COX-2 has also received much attention recently as it is involved in diseases associated with dysregulated inflammatory conditions, such as rheumatoid and osteoarthritis, cardiovascular disease, and the carcinogenesis process (16-20). COX-2 undergoes immediate-early up-regulation in response to an inflammatory stimulus (20, 21), such as a wound. It functions by producing prostaglandins that control many aspects of the resulting inflammation, including the induction of vascular permeability and the infiltration and activation of inflammatory cells (22). Interest in the role of the COX-2 pathway and other aspects of inflammation in the adult wound repair process is increasing (35) as these early events have been shown to regulate the outcome of repair. Based on the involvement of COX-2 in inflammation and the recent demonstration that it contributes to several aspects of adult wound repair (23-25), the role of COX-2 in the fetal wound healing process has been examined. These studies demonstrate differential expression of the COX-2 enzyme in early and late gestation fetal wounds.
Furthermore, PGE2, a COX-2 product shown to mediate many processes in the skin, caused a delay in healing and the production of a scar when introduced into early fetal wounds. The involvement of the COX-2 pathway in scar formation is further highlighted by the fact that increasing PGE2 levels in scarless wounds results in the conversion of a scarless healing process into one of repair with the generation of a scar. The introduction of PGE2 induced inflammation in fetal wounds (26), although their effect on collagen deposition or fibrosis was not examined. Whether PGE2 displays immunosuppressive or anti-inflammatory properties or instead acts as a pro-inflammatory molecule most likely results from differences in the expression or activity of the receptors for PGE2. There are several plausible mechanisms by which PGE2 could be inducing scar formation in fetal wounds. PGE2 could be enhancing acute inflammation, already known to interfere with scarless healing, thereby indirectly promoting scar formation through the recruitment and activation of inflammatory cells. PGE2 treatment could be both delaying healing and promoting scar tissue deposition through increases in the pro-fibrotic TGFβ (27). Disruption of the TGFβ signaling pathway in smad3-deficient mice has been shown to speed the rate of healing, and extensive data demonstrates restricted TGFβ3 levels are crucial to scarless healing. Lastly, there are data demonstrating increased fibroblast proliferation in response to PGE2 suggests that PGE2 could be directly stimulating fibroblasts to proliferate, amplifying collagen production and scarring. This idea is also supported by previous studies demonstrating an increase in collagen deposition and proliferation by fibroblasts following exposure to PGE2. The substantial data suggested the low levels of COX-2 expression and PGE2 may be necessary for the scarless repair of fetal skin. The fact that PGE2 induces scar formation in fetal skin further supports a role for the COX-2 pathway in scar formation. Using a COX-2 inhibitor celecoxib to treat incisional wounds, the role of COX-2 in the wound healing process was examined with significant inhibition of several parameters of inflammation in the wound site (28). This decrease in the early inflammatory phase of wound healing had an effect on later events in the wound healing process, namely a reduction in scar tissue formation, without disrupting reepithelialization or decreasing tensile strength.
Multi-Targeted siRNA Compositions
RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene (33, 34). In naturally occurring RNA interference, a double stranded RNA is cleaved by an RNase III/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3′ ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC, and guides the complex towards a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it. Studies have revealed that the use of chemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammalian cells, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function (33, 36, 37).
Importantly, it is presently not possible to predict with high degree of confidence which of many possible candidate siRNA sequences potentially targeting an mRNA sequence of a disease gene will, in fact, exhibit effective RNAi activity. Instead, individually specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested in mammalian cell culture to determine whether the intended interference with expression of a targeted gene has occurred. The unique advantage of siRNA makes it possible to be combined with multiple siRNA duplexes to target multiple disease causing genes in the same treatment, since all siRNA duplexes are chemically homogenous with same source of origin and same manufacturing process (33, 36-40).
There is a pressing need to provide potent siRNA duplexes targeting the pro-inflammatory factor TGFβ1, the inflammation promoter COX-2, and the differentiation regulator HoxB1 for scarless wound healing of skin. There further is a need to formulate such siRNA duplexes into multi-targeted siRNA compositions. There further remains a need to provide a therapeutic approach to improve the healing results of patients suffering wounds caused by injury, surgery, and many diseases.
Histidine-Lysine Polymer (HKP) Nanoparticle for SiRNA Delivery In Vivo
Histidine-Lysine Polymer (HKP), a cationic branched polymer, has been used for plasmid DNA and siRNA delivery in vivo. Recently, we have used HKP for siRNA delivery in various tissue types, including tumor, ocular, brain, lung and joint. A pair of the HK polymer species, H3K4b and PT73, has a Lysine backbone with four branches containing multiple repeats of Histidine, Lysine or Asparagine. When this HKP aqueous solution was mixed with siRNA at a N/P ratio of 4:1 by mass, the nanoparticles (average size of 100-200 nm in diameter) were self-assembled.